A critical function for all living organisms is the ability to move when needed. These movements-- intracellular trafficking, cell division, muscle contraction, and cell motility-- are driven by molecular machines that exert an amazing amount of force considering that they are only a few nanometers across. Given the variety of motor proteins in the cell, a key question is how motors cooperate and compete while moving cargoes and applying forces. An emerging paradigm is the notion of specialized motors, or motors that are fine-tuned to perform a specific function. Despite the importance of these motor proteins, relatively little is known about their individual adaptations and how these relate to the motility patterns found in the cell. In prior studies, we discovered two myosins with new and distinct processive stepping patterns on actin. Myosin X walks along multiple filaments in a fascin-actin bundle. Nonmuscle myosin IIB (NMIIB), on the other hand, walks along the long-pitch helix of a single actin filament. Both myosins play pivotal roles in migrating cells, including metastasizing tumor cells. Myosin X delivers essential cargoes such as integrins, cadherins and netrin receptors to filopodia at the leading edge of the cell; NMIIB appears in the rear of the cell, wher it maintains cell polarity and internal organization. Thus, it is essential that we understand how both myosins operate so that we can control cell motility through these players. For both myosins, we propose that their two motor domains are synchronized through strain-sensitive gating mechanisms. For both NMIIB and myosin X, gating is tuned to accommodate their unique stepping patterns along actin tracks. We hypothesize that NMIIB has adaptations for tension maintenance, myosin X has adaptations for bundle-selection, and both may have adaptations for twisting single actin filaments. To test these gating mechanisms, we will pursue the following specific aims: Aim 1: We will determine how NMIIB takes short, processive steps along actin and maintains cytoskeletal tension. Aim 2: We will determine how myosin X is gated in the environment of an actin filament bundle. Aim 3: We will determine how both myosins twist actin filaments. PUBLIC HEALTH RELEVANCE: Motile cells use the motor activity of nonmuscle myosin IIB and myosin X to organize and reposition their contents. We will use a battery of advanced single-molecule methods, as well as the tools of molecular genetics, to determine how these myosins are coordinated on their distinct types of actin tracks. Together this work will illuminate how cancer cells escape from the primary tumor, migrate, and invade surrounding tissues.